Hi- My institution is interested in acquiring a CyTOF (Fluidigm Hyperion) for mass cytometry and imaging applications. I have seen multiple polished presentations on the technology and I would be interested to chat with any users in the field particularly from a…

Hi-
My institution is interested in acquiring a CyTOF (Fluidigm Hyperion) for mass
cytometry and imaging applications. I have seen multiple polished presentations
on the technology and I would be interested to chat with any users in the field
particularly from a core facility background (eg flow core or MS core) that are
willing to share their experiences with this. I am most interested to hear
about the sample prep/conjugation process and experiences on how well this has
been implemented in facility work. Please contact me online or offline, your
preference. Any shared experiences would be greatly appreciated.
Scott-
Scott Shaffer, Ph.D.
Director, Mass Spectrometry Facility
Professor, Biochemistry and Molecular Pharmacology
University of Massachusetts Medical School
222 Maple Ave / Fuller Building
Shrewsbury, MA 01545
508.856.8917
<email obscured><email obscured>>

To elaborate on Brian's informative response: I presented a poster at ABRF 2003 on the consequences of adding various volumes of acetonitrile to serum, per the following link: http://www.polylc.com/Downloads/ABRF_2003_SEC_poster.pdf If the ratio of acetonitrile to serum was 1:1, then peptides < 25…

To elaborate on Brian's informative response:
I presented a poster at ABRF 2003 on the consequences of adding various volumes
of acetonitrile to serum, per the following link:
http://www.polylc.com/Downloads/ABRF_2003_SEC_poster.pdf
If the ratio of acetonitrile to serum was 1:1, then peptides < 25 kDa remained
in solution while anything larger precipitated. If the ratio was 2:1, then
only peptides < 6 kDa remained in solution, etc. This work was followed up in
the following two papers:
1) K. Merrell et al., J. Biomolec. Techniques 15 (2004) 238;
2) O. Chertov et al., Expert Rev. Proteomics 2 (2005) 139.
The Chertov review in particular is quite thorough. They demonstrated that
substituting propanol for acetonitrile resulted in larger peptides remaining in
solution (up to 15 kDa instead of 6 kDa with a 2:1 ratio, etc.).
This approach will permit you to fractionate large volumes of serum.
Andy Alpert
PolyLC Inc.
<email obscured>

Hello, I have a question about initial analytical process on serum. I would like to do enrichment of peptides and small molecular weight proteoforms from serum. One of my idea, will be to use solid phase extraction (SPE) cartridge as C18. Could…

Hello,
I have a question about initial analytical process on serum. I would like to do
enrichment of peptides and small molecular weight proteoforms from serum. One
of my idea, will be to use solid phase extraction (SPE) cartridge as C18.
Could you please suggest me some reference using SPE or chromatographic phase
on serum to isolate some target analytes from serum?
Thanks a lot!

Hello Luc, I am not certain if your proposal to use C18 would be your first step to enrich for small molecular weight peptides. If so I would not do that as the C18 will bind all peptides and proteins regardless of…

Hello Luc,
I am not certain if your proposal to use C18 would be your first step to enrich
for small molecular weight peptides. If so I would not do that as the C18 will
bind all peptides and proteins regardless of size.
It is common to use organic solvents to crash out proteins, then take the
supernatant after spinning out the insoluble proteins and work with that to
isolate your peptides and small molecules etc. You could take this supernatant
and apply directly to an SCX cartridge in acid conditions to bind everything
with a + charge, or evaporate the organic solvent and apply that to your C18
cartridge then elute with an organic solvent.
- You can modify the conditions of organic precipitation in hopes of being
selective for those things you are looking for.
- If you need native conditions to isolate the analytes of interest, you could
use a size exclusion column and pool the lower MW fractions.
- You could use ammonium sulfate precipitation and use C18 to capture the
material that is still in the supernatant.
In any event your first step should be to separate your analytes of interest
from the bulk of the proteins in the serum and then isolate your analytes from
this protein depleted fraction.
Cheers,
Brian
> On Mar 13, 2018, at 7:11 AM, Abrf Proteome <email obscured>> wrote:
>
> Hello,
>
> I have a question about initial analytical process on serum. I would like to
do enrichment of peptides and small molecular weight proteoforms from serum .
One of my idea, will be to use solid phase extraction (SPE) cartridge as C18.
> Could you please suggest me some reference using SPE or chromatographic phase
on serum to isolate some target analytes from serum?
>
> Thanks a lot!
>
> Luc
>
>> Le 1 mars 2018 à 21:18, Hampton, Brian <email obscured>> a écrit
:
>>
>> Hi Achim,
>>
>> Thanks for pointing out this method!
>>
>> Brian
>>
>>> On Mar 1, 2018, at 12:24 PM, Achim <email obscured>> wrote:
>>>
>>>
>>> There is an interesting thread with a discussion of the S-trap method and
references to Alexandre Zougman's paper
(http://onlinelibrary.wiley.com/doi/10.1002/pmic.201300553/abstract) on the
SharedProteomics discussion forum
(http://sharedproteomics.com/forum/viewtopic.php?t=2942)

Hi Luc, I worked for many years in the Lower Saxony Institute for Peptide Research, focusing on peptides from human hemofiltrate. This was generated using a 20 kDa cutoff filter membrane to clear the blood of final renal disease patients from toxic…

Hi Luc,
I worked for many years in the Lower Saxony Institute for Peptide Research,
focusing on peptides from human hemofiltrate. This was generated using a 20 kDa
cutoff filter membrane to clear the blood of final renal disease patients from
toxic compounds. We looked at the peptides in there and found uncountable
numbers of peptides, mainly fragments of larger proteins. But some of them had
been very interesting, but due to some scientific miss management we could not
follow up. These had been beta-defensin, endostatin and many more, forgot the
names after 19 years.
Try a 20 kDa cutoff spin filter, SPE will not help to isolate smaller peptides,
wash the retained serum a few times and concentrate the flow through with SPE
finally.
Manfred
﻿Am 13.03.18, 19:11 schrieb "ABRF Discussion Forum im Auftrag von Abrf
Proteome" <email obscured> im Auftrag von <email obscured>>:
Hello,
I have a question about initial analytical process on serum. I would like
to do enrichment of peptides and small molecular weight proteoforms from serum
. One of my idea, will be to use solid phase extraction (SPE) cartridge as C18.
Could you please suggest me some reference using SPE or chromatographic
phase on serum to isolate some target analytes from serum?
Thanks a lot!
Luc
> Le 1 mars 2018 à 21:18, Hampton, Brian <email obscured>> a
écrit :
>
> Hi Achim,
>
> Thanks for pointing out this method!
>
> Brian
>
>> On Mar 1, 2018, at 12:24 PM, Achim <email obscured>>
wrote:
>>
>>
>> There is an interesting thread with a discussion of the S-trap method
and references to Alexandre Zougman's paper
(http://onlinelibrary.wiley.com/doi/10.1002/pmic.201300553/abstract) on the
SharedProteomics discussion forum
(http://sharedproteomics.com/forum/viewtopic.php?t=2942)
>> ――
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